Elevated Nitrite/Nitrate Ratio as a Potential Biomarker for the Differential Diagnosis of Pleural Effusions.

Pleural effusions (PEs) are common in clinical practice and can be due to many different underlying diseases such as cancer, congestive heart failure, or pneumonia. An accurate differential diagnostic categorization is essential, as the treatment and prognosis of PEs largely depend on its cause. In this study, we tested the hypothesis that nitrite and nitrate concentrations in PEs are associated with the inflammation and infection conditions. We therefore measured the nitrite and nitrate levels in 143 PE samples using a sensitive liquid chromatography-tandem mass spectrometry method and investigated their diagnostic potential in differentiating PEs. The results showed that nitrite concentrations and nitrite/nitrate ratios were higher in exudates than in transudates (NO2-: 2.12 vs. 1.49 µM; NO2-/NO3-: 23.3 vs. 14.0). Both the nitrite concentrations and the nitrite/nitrate ratios were positively correlated with the three Light's criteria. Moreover, the receiver operating characteristic curve analysis revealed that the nitrite/nitrate ratio with an area under the curve of 0.71 could be a potential diagnostic biomarker in separating infectious PEs (IPEs) from other types of PEs. Taken together, the nitrite/nitrate ratio not only reflected the statuses of inflammation, but also the nitrate reduction by pathogenic bacteria infection in the pleural cavity. The nitrite/nitrate ratio could be a better biomarker in the differential diagnosis of PEs than the nitrite concentration alone.

Alkylating and oxidative stresses in smoking and non-smoking patients with COPD: Implications for lung carcinogenesis.

Chronic obstructive pulmonary disease (COPD) is a disease characterized by chronic inflammation and irreversible airway obstruction. Cigarette smoking is the predominant risk factor for developing COPD. It is well-known that the COPD is also strongly associated with an increased risk of developing lung cancer. Cigarette smoke contains elevated concentrations of oxidants and various carcinogens (e.g., tobacco-derived nitrosamines) that can cause oxidative and alkylating stresses, which can also arise from inflammation. However, it is surprising that, except for oxidative stress, little information is available on the burden of alkylating stress and the detoxification efficiency of tobacco-derived carcinogens in COPD patients. In this study, we used LC-MS/MS to measure the archetypical tobacco-specific carcinogenic 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), its major metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), three biomarkers of oxidative stress (8-oxo-7,8-dihydroguanine, 8-oxoGua; 8-oxo-7,8-dihydro-2'-deoxyguanosine, 8-oxodGuo; 8-oxo-7,8-dihydroguanosine, 8-oxoGuo) and two biomarkers of alkylating stress (N7-methylguanine, N7-MeGua and N3-methyladenine, N3-MeAde), in the urine of smoking and non-smoking COPD patients and healthy controls. Our results showed that not only was oxidative stress significantly elevated in the COPD patients compared to the controls, but also alkylating stress. Significantly, levels of alkylating stress (i.e., N7-MeGua) were highly correlated with the COPD severity and not affected by age and smoking status. Furthermore, COPD smokers had significantly higher ratios of free NNAL to the total NNAL than control smokers, implying a lower detoxification efficiency of NNK in COPD smokers. This ratio was even higher in COPD smokers with stages 3-4 than in COPD smokers with stages 1-2. Taken together, our results demonstrated that the detoxification efficiency of tobacco-derived carcinogens (e.g., NNK) was associated with the pathogenesis and possibly the progression of COPD. In addition to oxidative stress, alkylating stress derived from chronic inflammation appears to be also dominant in COPD patients.

Clinical relevance of guanine-derived urinary biomarkers of oxidative stress, determined by LC-MS/MS.

A reliable and fast liquid chromatography-tandem mass spectrometry method has been developed for the simultaneous determination of three oxidized nucleic acid damage products in urine, 8-oxoguanine (8-oxoGua), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo). We applied this method to assess the effect of various urine workup procedures on the urinary concentrations of the oxidized nucleic acid products. Our results showed that frozen urine samples must be warmed (i.e., to 37 °C) to re-dissolve any precipitates prior to analysis. We showed that common workup procedures, such as thawing at room temperature or dilution with deionized water, are not capable of releasing fully the oxidized nucleic acid products from the precipitates, and result in significant underestimation (up to ~ 100% for 8-oxoGua, ~ 86% for both 8-oxodGuo and 8-oxoGuo). With this method, we further assessed and compared the ability of the three oxidized nucleic acid products, as well as malondialdehyde (MDA, a product of lipid peroxidation), to biomonitor oxidative stress in vivo. We measured a total of 315 urine samples from subjects with burdens of oxidative stress from low to high, including healthy subjects, patients with chronic obstructive pulmonary disease (COPD), and patients on mechanical ventilation (MV). The results showed that both the MV and COPD patients had significantly higher urinary levels of 8-oxoGua, 8-oxodGuo, and 8-oxoGuo (P < 0.001), but lower MDA levels, compared to healthy controls. Receiver operating characteristic curve analysis revealed that urinary 8-oxoGuo is the most sensitive biomarker for oxidative stress with area under the curve (AUC) of 0.91, followed by 8-oxodGuo (AUC: 0.80) and 8-oxoGua (AUC: 0.76). Interestingly, MDA with AUC of 0.34 failed to discriminate the patients from healthy controls. Emerging evidence suggests a potential clinical utility for the measurement of urinary 8-oxoGuo, and to a lesser extent 8-oxodGuo, which is strongly supported by our findings.

Elevated urinary levels of carcinogenic N-nitrosamines in patients with urinary tract infections measured by isotope dilution online SPE LC-MS/MS.

N-nitrosamines (NAms) are well-documented for their carcinogenic potential. Human exposure to NAms may arise from the daily environment and endogenous formation via the reaction of secondary amines with nitrites or from bacteria infection. We describe the use of isotope dilution online solid-phase extraction (SPE) LC-MS/MS to quantify nine NAms in human urine. This method was validated and further applied to healthy subjects and patients with urinary tract infection (UTI). N-nitrosodimethylamine (NDMA), N-nitrosomethylethylamine (NMEA), N-nitrosopyrrolidine (NPYR) and N-nitrosomorpholine (NMOR) were analyzed with an APCI source, while N-nitrosodiethylamine (NDEA), N-nitrosopiperidine (NPIP), N-nitrosodi-n-propylamine (NDPA), N-nitrosodibutylamine (NDBA) and N-nitrosodiphenylamine (NDPhA) were quantified with an ESI source, due to their effect on the sensitivity and chromatography. NDMA was the most abundant N-nitrosamine, while NDPhA was firstly identified in human. UTI patients had three to twelve-fold higher concentrations for NDMA, NPIP, NDEA, NMOR and NDBA in urine than healthy subjects, and the NAms were significantly decreased after antibiotics treatment. NDMA concentrations were also significantly correlated with the pH value, leukocyte esterase activity or nitrite in urines of UTI patients. Our findings by online SPE LC-MS/MS method evidenced that UTI patients experienced various NAms exposures, especially the potent carcinogen NDMA, which was likely induced by bacteria infection.

Urinary nitrite/nitrate ratio measured by isotope-dilution LC-MS/MS as a tool to screen for urinary tract infections.

Urinary tract infections (UTIs) are the most common type of nosocomial infection. Traditionally, the presence of white blood cells and microorganisms in the urine provides objective evidence for UTI diagnosis. Here, we describe the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to measure the nitrite and nitrate levels in urine and investigate the potential of this method for UTI diagnosis. LC-MS/MS analysis was performed in positive electrospray ionization mode. After adding (15)N-labeled internal standards and derivatizing with 2,3-diaminonaphthalene (DAN), the urinary nitrite content was directly analyzed by LC-MS/MS, whereas the urinary nitrate was first reduced to nitrite before derivatization and LC-MS/MS analysis. The derivatization of nitrite and enzymatic reduction of nitrate were optimized. This method was then applied to 241 healthy subjects and 73 UTI patients. Optimization tests revealed that 1 mL of crude urine required at least 6.25 µmol of DAN to completely derivatize nitrite and 2.5 U of nitrate reductase to completely reduce nitrate to nitrite. Urinary analysis showed that the urinary concentration of nitrite and the nitrite/nitrate ratio were higher in UTI patients than in healthy subjects. Compared with the dipstick-based urinary nitrite test and using LC-MS/MS to determine the nitrite concentration (sensitivity: 23-25%), the nitrite/nitrate ratio was significantly more sensitive (95%) and exhibited a satisfactory specificity (91%) in the screening of UTIs. Taken together, the nitrite/nitrate ratio, which reflects the reducing ability of pathogenic bacteria, could be a better method for the diagnosis of UTIs that is not subject to variations in urine specimen quality.